Acetyl-CoA is an extremely important intermediate in metabolic pathways of microorganisms. Various metabolites are produced via acetyl-CoA. Known examples of such substances produced via acetyl-CoA include amino acids such as L-glutamic acid, L-glutamine, L-proline, L-arginine, L-leucine and L-isoleucine; organic acids such as acetic acid, propionic acid, butyric acid, caproic acid, citric acid, 3-hydroxybutyric acid, 3-hydroxyisobutyric acid, 3-aminoisobutyric acid, 2-hydroxyisobutyric acid, methacrylic acid and poly-3-hydroxybutyric acid; alcohols such as isopropyl alcohol, ethanol and butanol; acetone; and polyglutamic acid.
L-Glutamic acid, or 2-aminopentanedioic acid, is a naturally occurring amino acid and a constituent of fermented foods like soy sauce, fish sauce, fermented bean paste and cheese. The sodium salt of glutamic acid, known as monosodium glutamate or MSG, is widely used in the food industry as a seasoning agent.
A promising candidate for industrial glutamic acid production is the Gram negative bacterium belonging to the genus Pantoea from the Enterobacteriaceae family. In addition to producing high glutamic acid titers, the bacterium Pantoea ananatis is also resistant to high concentrations of glutamic acid and can grow at acidic pH (Appl. Microbiol. Biotechnol. 93:331-341 (2012)). With the establishment of genetic manipulation techniques for P. ananatis, superior recombinant strains have been successfully developed that demonstrate substantially higher glutamic acid production capacities (U.S. Pat. No. 6,331,419B1, U.S. Pat. No. 7,015,010B1).
In P. ananatis, a carbon source like glucose is metabolized via the Embden-Meyerhof-Parnas pathway to form pyruvate. Subsequently, acetyl-CoA is formed from pyruvate by the action of pyruvate dehydrogenase and/or pyruvate formate lyase with the concomitant loss of valuable carbon derived from the sugar in the form of by-products like carbon dioxide and/or formate. Thereafter, glutamic acid is produced from acetyl-CoA via the Krebs cycle intermediate 2-oxoglutarate. Naturally, in this pathway, the maximum yield of glutamic acid that can be achieved is limited by the inherent loss of carbon as carbon dioxide and thus, engineering an alternate pathway that can circumvent this loss and/or cause the fixation of carbon dioxide into the pathway can help to improve the overall yield, efficiency and economics of the fermentation process.
There are several known pathways in which carbon dioxide is fixed to provide a carbon source in microorganisms (Appl. Environ. Microbiol. 77(6), 1925-1936(2011)). Specific examples of the pathways include the Calvin-Benson cycle, reductive TCA cycle, Wood-Ljungdahl pathway, 3-hydroxypropionate cycle and 4-hydroxybutyrate cycle. The Calvin-Benson cycle is a CO2 fixation pathway existing in plants and photosynthetic bacteria, and composed of about 12 kinds of enzymes, wherein CO2 is fixed by ribulose-1,5-bisphosphate carboxylase (RubisCO) to produce glyceraldehyde 3-phosphate. The reductive TCA cycle is a cycle found in anaerobic bacteria and microaerophilic bacteria including green sulfur bacteria, and composed of 11 kinds of enzymes. The cycle is characterized by CO2 fixation enzymes using ferredoxin as a coenzyme (acetyl-CoA carboxylase, 2-oxoglutarate synthase), and pyruvate is produced from CO2 by a reaction in the reverse direction of the normal TCA cycle. The Wood-Ljungdahl pathway is a pathway found in anaerobic microorganisms such as acetic acid-producing bacteria, and composed of 9 kinds of enzymes, wherein CO2 and formate on a coenzyme are reduced by formate dehydrogenase, CO dehydrogenase and the like to finally achieve conversion to acetyl-CoA. The 3-hydroxypropionate cycle is found in Chloroflexus bacteria and the like, and composed of 13 kinds of enzymes, wherein CO2 is fixed by the action of acetyl-CoA (propionyl-CoA) carboxylase and acetyl-CoA is produced via malonyl-CoA and the like. The 4-hydroxybutyrate cycle is a pathway existing in archaebacteria and the like. In this cycle, CO2 is fixed by the reactions of pyruvate synthase, acetyl-CoA (propionyl-CoA) carboxylase and phosphoenolpyruvate carboxylase, and acetyl-CoA is produced via 4-hydroxybutyryl-CoA and the like.
U.S. Pat. No. 7,785,858B2 patent document describes the engineering of an alternate pathway for acetyl-CoA formation with reduced production of carbon dioxide by enhancing the activity of the Bifidum pathway enzymes D-xylulose-5-phosphate phosphoketolase and/or fructose-6-phosphate phosphoketolase. As a result, theoretically, 1 mole of CO2 can be saved per mole of glucose that is consumed via this alternative route. When this pathway was introduced to a glutamic acid-producing strain of P. ananatis, the glutamic acid titer increased by 2.6 g/L which corresponded to a 6% increase in overall glutamic acid yield.
On the other hand, several reports have been made as ideas attempting to introduce a pathway for fixation of carbon dioxide to a useful-compound-producing microorganism in order to produce a useful substance. For example, WO2009/094485 and WO2010/071697 disclose proposals to use a microorganism to which a pathway similar to the Wood-Ljungdahl pathway in acetic acid bacteria was introduced, in order to produce acetyl-CoA from carbon dioxide. Further, as an example of production of a useful compound by fixation of CO2, WO2009/046929 discloses an attempt to use a microorganism to which hydrogenase and tetrahydrofolate lyase were introduced, in order to produce lactic acid from carbon dioxide. Further, WO2011/099006 proposes a cycle in which CO2 is fixed via a reaction for fixation of carbon dioxide on acetyl-CoA and a reduction reaction of malonyl-CoA. DE 102007059248 A proposes production of acetyl-CoA by a pathway similar to the 4-hydroxybutyrate cycle.